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A brief resume of the intended work: Need of study


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a. BRIEF RESUME OF THE INTENDED WORK:

Need of study:

Wounds are may arise due to physical injury, chemical injury or microbial infections. Healing of wounds usually takes place in a direction away from its normal course and under-healing, over- healing or no healing of wound is common. Treatment of under healing of wounds and prevention wound infection is a very expensive and complicated program. Several drugs from plants are known to possess wound healing activity. Some of these plants have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients, but the potential of most remains unexplored1. The best known wound healing agents from plant source are Aloe vera and Centella asiatica2,3. The search of a potent wound healing agent from plant source is continuing and different plants used traditionally in the treatment of wounds are being screened for wound healing activity. Recently, we reported that methanolic extract of Tectona grandis leaves used traditionally in Mangalore region of Karnataka possess good wound healing activity4.

One of the plants traditionally used for treatment of wounds in Andhra Pradesh is Bauhinia purpurea. The leaves of the plant are made into a paste and applied over the wounds. It is believed that the plant leaves helps in faster healing of wounds.

The present study will be undertaken to evaluate the effect of chloroform and methanolic extracts of Bauhinia purpurea leaves on experimentally induced wounds in rats.



Review of Literature:

The plant Bauhinia purpurea (Fabaceae) is native to South China (which includes Hong Kong), Southeastern Asia and United States of America5. In India, the leaves of the plant are used mainly as plates for serving meal. Isolated compounds from the bark of the plant are reported to possess antimycobacterial, antimalarial, antifungal, cytotoxic, and anti-inflammatory activities6. Bauhiniastatins isolated from leaves and bark of the plant were found to inhibit human cancer cell lines7. Bauhinia purpurea bark extract is reported to increase the serum levels triiodothyronine (T3) and thyroxine (T4)8. The lectins isolated from the plant are used as markers for the diagnosis of pneumonitis, fibrosis and different types of cancer in various organs9-14.

The chemical composition of Bauhinia purpurea has been well studied. Eleven new secondary metabolites (1-11), together with two known flavanones (12 and 13) and five known bibenzyls (14-18) have been isolated from the root extract of Bauhinia purpurea, which includes eight dihydrodibenzoxepins (1-8), a dihydrobenzofuran (9), a novel spirochromane-2,1'-hexenedione (10), and a new bibenzyl (11)5. Bioassay-guided (P388 lymphocytic leukemia cell line) separation of extracts prepared from the leaves, stems, and pods of Bauhinia purpurea, and, in parallel, its roots, led to the isolation of four new dibenz[b,f]oxepins (2a, 3-5) named bauhiniastatins 1-4, as well as the known and related pacharin (1) as cancer cell growth inhibitors6.

Objective of study:

The objective of the proposed study is to evaluate the wound healing activity of chloroform and methanolic extracts of Bauhinia purpurea leaves on experimentally induced wounds in rats.



SPECIFIC OBJECTIVES:

  • To study the effect of chloroform and methanolic extracts of Bauhinia purpurea on incision wound and excision wound in rats.

  • To evaluate the effect of chloroform and methanolic extracts of Bauhinia purpurea in burn wound model in rats.

  • To study the effect of oral administration of chloroform and methanolic extracts of Bauhinia purpurea in dead space wound model.

  • To determine the skin toxicity of chloroform and methanolic extracts of Bauhinia purpurea in rabbits.

b. MATERIALS AND METHODS:

Source of Data:

Data will be obtained from CD-Rom, Internet facilities, Literatures and related articles from libraries of Krupanidhi College of Pharmacy, Indian Institute of Sciences, Government College of Pharmacy etc., and other Research Publications and Journals.



Method of Collection of Data:

The data collected will be based on animal experimentation as per the parameters studied under each animal model, which are mentioned under the objectives of the study.



Methods:

The animals will be grouped as follows

Group I: Control

Group II: Standard drug (Aloe vera 5% for local application and 300 mg/kg p.o)15

Group III: Low dose of chloroform extract of Bauhinia purpurea

Group IV: High dose of chloroform extract of Bauhinia purpurea

Group V: Low dose of methanolic extract of Bauhinia purpurea

Group VI: High dose of methanolic extract of Bauhinia purpurea

The low dose of the extracts will be 2.5% w/w in suitable base and high dose will be 5% in suitable base for local application. The dose for oral administration in dead space wound model will be selected after acute toxicity study using OPPTS limit test16.


  1. Preparation of extracts and preliminary phytochemical investigation: The leaves of the plant will be purchased from local market and the dried leaves will be subjected to Soxhlation. Initially, the leaves will be defatted using petroleum ether followed by successive extractions with chloroform and methanol.

  2. Preparation of suitable formulation: The methanolic extract formulation will be prepared using hydrophobic base and chloroform extract will be formulated using hydrophilic base17.

  3. Effect on excision and incision wound: The excision wound will be induced by excising 500 mm2 of the skin in the dorsal thoracic region of the rat. Drug will be applied locally. The wound area and the falling of scar will be taken as parameters for determination of wound healing18,19. The incision wound will be created by cutting 6 cm length of entire skin on either side of the vertebral column, the breaking strength of the wound will be determined using constant water flow technique after 10 days of local application of drugs20-22.

  4. Effect on burn wound: Partial thickness burn wounds were inflicted under pentobarbitone (30 mg/kg, i.p.) anesthesia by pouring hot molten wax at 80 0C. The wax was poured on the shaven back of the animal through a cylinder of 300 mm2 circular opening. The parameters will be same as those mentioned under excision wound model23,24.

  5. Effect of dead space wound: This type of wound will be created by implanting subcutaneously a 2.5x0.5 cm polypropylene tube in the lumber region of dorsal side in anesthetized rats. Drugs will be administered orally for 10 days. The breaking strength of the granulation tissue formed over the tube and the hydroxyproline content in the granulation tissue will be determined25.

  6. Skin irritation study: This will be done using rabbits. The formulation of will be applied to the shaved skin on the back of the rabbit. After 4 hr, the skin will be observed for signs of inflammation and scores will be given based on the severity of inflammation26.

Does the study require any investigation or interventions to be conducted on patients or the human or animals? If so please describe briefly:

YES


Study requires investigation on animals. The effects of the drug will be studied on various parameters using rats and rabbits as experimental animals.

Has ethical clearance been obtained from your institute

Ethical Committee approval letter is enclosed along with the hard copy



c. LIST OF REFERENCES:

  1. Biswas TK, Mukherjee B. Plant medicines of Indian origin for wound healing activity: a review. Int J low Extrem Wounds 2003;2(1): 25-39.

  2. Heggers JP, Kucukcelebi A, Listengarten D, Stabenau J, Ko F, Broemeling LD, Robson MC, Winters WD. Beneficial effect of Aloe on wound healing in an excisional wound model. J Altern Complement Med 1996 Summer;2(2):271-7.

  3. Shukla A, Rasik AM, Jain GK, Shankar R, Kulshrestha DK, Dhawan BN. In vitro and in vivo wound healing activity of asiaticoside isolated from Centella asiatica. J Ethnopharmacol 1999 Apr;65(1):1-11

  4. Majumdar M, Nayeem N, Kamath JV, Asad M. Evaluation of Tectona grandis leaves for wound healing activity. Pak J Pharm Sci 2007 Apr;20(2):120-4.

  5. http://en.wikipedia.org/wiki/Bauhinia_purpurea. Retrieved on 21/09/2007 , 11.30 AM IST.

  6. Boonphong S, Puangsombat P, Baramee A, Mahidol C, Ruchirawat S, Kittakoop P. Bioactive compounds from Bauhinia purpurea possessing antimalarial, antimycobacterial, antifungal, anti-inflammatory, and cytotoxic activities. J Nat Prod 2007 May;70(5):795-801.

  7. Pettit GR, Numata A, Iwamoto C, Usami Y, Yamada T, Ohishi H, Cragg GM. Antineoplastic agents. 551. Isolation and structures of bauhiniastatins 1-4 from Bauhinia purpurea. J Nat Prod 2006 Mar;69(3):323-7.

  8. Panda S, Kar A. Withania somnifera and Bauhinia purpurea in the regulation of circulating thyroid hormone concentrations in female mice. J Ethnopharmacol 1999 Nov 1;67(2):233-9

  9. Sarker AB, Koirala TR, Murakami I, Jeon HJ. Bauhinia purpurea and Pisum sativum lectin binding in human breast. Indian J Pathol Microbiol 1995 Jul;38(3):261-5

  10. Sarker AB, Akagi T, Teramoto N, Nose S, Yoshino T, Kondo E.
    Bauhinia purpurea (BPA) binding to normal and neoplastic thyroid glands. Pathol Res Pract 1994 Nov;190(11):1005-11.

  11. Kasper M, Schuh D, Müller M. Bauhinia purpurea lectin (BPA) binding of rat type I pneumocytes: alveolar epithelial alterations after radiation-induced lung injury. Exp Toxicol Pathol 1994 Oct;46(4-5):361-7

  12. Sarker AB, Koirala TR, Murakami I. Bauhinia purpurea agglutinin (BPA) binding sites in human gastrointestinal tract. Indian J Pathol Microbiol 1994 Jan;37(1):21-8

  13. Shue GL, Kawa S, Kato M, Oguchi H, Kobayashi T, Koiwai T, Tokoo M, Furuta S, Kanai M, Homma T. Expression of glycoconjugates in pancreatic, gastric, and colonic tissue by Bauhinia purpurea, Vicia villosa, and peanut lectins. Scand J Gastroenterol 1993 Jul;28(7):599-604

  14. Yamamoto K, Konami Y, Kusui K, Osawa T. Purification and characterization of a carbohydrate-binding peptide from Bauhinia purpurea lectin. FEBS Lett 1991 Apr 9;281(1-2):258-62.

  15. Rajasekaran S, Sriram N, Arulselvan P, Subramanian S. Effect of Aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes. Pharmazie 2007 Mar;62(3):221-5.

  16. Health Effect Test Guidelines. Acute Oral Toxicity (Computer programme OPPTS 870.1100 United State Office of Prevention. Pesticides and Toxic Substances Environmental Protection Agency (7101). Available from URL: http://www.epa.gov/oppts/home/guideline.htm.

  17. Cooper and Gunn’s Dispencing for Pharmaceutical Students, Gunn C, Carter SJ (Eds), CBS publishers & distributors, New Delhi, India, 1995, pp 158-161

  18. Kamath JV, Rana AC, Chowdhury AR. Pro-healing effect of Cinnamomum zeylanicum bark. Phytother Res 2003;17: 970-972.

  19. Morton JJ, Malone MH. Evaluation of vulnerary activity by open wound procedure in rats. Arch Int Pharmacodyn Ther 1972;196: 117-126.

  20. Lee KH. Studies on the mechanism action of salicylates II, effect of vitamin A on wound healing retardation action of aspirin. J Pharmacol Sci 1968;57: 1238-1240.

  21. Ehrich HP, Hunk TK. Effect of cortisone and anabolic steroids on tensile strength of healing wound. Ann Surg 1969;170: 203-206.

  22. Somayaji SN, Jacob AP, Bairy KL. Effect of tolmetin and its copper complex on wound healing. Indian J Exp Biol 1995;33(3):201-204.

  23. Holla RK, Sequeria RP, Kulkarni DR. Cylosporin and wound healing. Indian J Exp Biol 1998;26: 869-873.

  24. Rao CM, George KM, Bairy KL, Somayaji SN. An apprantial of the healing profiles of oral and external (gel) Metronidazole on partial thickness Burn wounds. Indian J Pharmacol 2000;32: 282-287.

  25. Padmaja PN, Bairy KL, Kulkarni DR. Pro healing effect of betel nut and its polyphenols. Fitoterapia 1994;LXV(4): 298-303.

  26. Gfeller W, Kobel W, Seifert G. Overview of animal test methods from skin irritation. Food Chem Toxicol 1985;23(2): 165-168.


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